Aav4 vector and uses thereof

ABSTRACT

The present invention provides an adeno-associated virus 4 (AAV4) virus and vectors and particles derived therefrom. In addition, the present invention provides methods of delivering a nucleic acid to a cell using the AAV4 vectors and particles.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/719,671 filed Mar. 8, 2010, which is a continuation of U.S. patentapplication Ser. No. 10/719,311 filed Nov. 20, 2003, which is acontinuation of now abandoned U.S. patent application Ser. No.09/254,747 filed Nov. 26, 1999, which is a national stage applicationunder 35 U.S.C. 371 of PCT Patent Application No. PCT/US97/16266 havingan international filing date of Sep. 11, 1997, which claims the benefitof Provisional Patent Application No. 60/025,934 filed Sep. 11, 1996,the entire disclosure of each of which are hereby incorporated herein byreference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention provides adeno-associated virus 4 (AAV4) andvectors derived therefrom. Thus, the present invention relates to AAV4vectors for and methods of delivering nucleic acids to cells ofsubjects.

2. Background Art

Adeno associated virus (AAV) is a small nonpathogenic virus of theparvoviridae family (for review see 28). AAV is distinct from the othermembers of this family by its dependence upon a helper virus forreplication. In the absence of a helper virus, AAV may integrate in alocus specific manner into the q arm of chromosome 19 (21). Theapproximately 5 kb genome of AAV consists of one segment of singlestranded DNA of either plus or minus polarity. The ends of the genomeare short inverted terminal repeats which can fold into hairpinstructures and serve as the origin of viral DNA replication. Physically,the parvovirus virion is non-enveloped and its icosahedral capsid isapproximately 20 nm in diameter.

To date 7 serologically distinct AAVs have been identified and 5 havebeen isolated from humans or primates and are referred to as AAV types1-5 (1). The most extensively studied of these isolates is AAV type 2(AAV2). The genome of AAV2 is 4680 nucleotides in length and containstwo open reading frames (ORFs). The left ORF encodes the non-structuralRep proteins, Rep40, Rep 52, Rep68 and Rep 78, which are involved inregulation of replication and transcription in addition to theproduction of single-stranded progeny genomes (5-8, 11, 12, 15, 17, 19,21-23, 25, 34, 37-40). Furthermore, two of the Rep proteins have beenassociated with the preferential integration of AAV genomes into aregion of the q arm of human chromosome 19. Rep68/78 have also beenshown to possess NTP binding activity as well as DNA and RNA helicaseactivities. The Rep proteins possess a nuclear localization signal aswell as several potential phosphorylation sites. Mutation of one ofthese kinase sites resulted in a loss of replication activity.

The ends of the genome are short inverted terminal repeats which havethe potential to fold into T-shaped hairpin structures that serve as theorigin of viral DNA replication. Within the ITR region two elements havebeen described which are central to the function of the ITR, a GAGCrepeat motif and the terminal resolution site (trs). The repeat motifhas been shown to bind Rep when the ITR is in either a linear or hairpinconformation (7, 8, 26). This binding serves to position Rep68/78 forcleavage at the trs which occurs in a site- and strand-specific manner.In addition to their role in replication, these two elements appear tobe central to viral integration. Contained within the chromosome 19integration locus is a Rep binding site with an adjacent trs. Theseelements have been shown to be functional and necessary for locusspecific integration.

The AAV2 virion is a non-enveloped, icosahedral particle approximately25 nm in diameter, consisting of three related proteins referred to asVP1, 2 and 3. The right ORF encodes the capsid proteins, VP1, VP2, andVP3. These proteins are found in a ratio of 1:1:10 respectively and areall derived from the right-hand ORF. The capsid proteins differ fromeach other by the use of alternative splicing and an unusual startcodon. Deletion analysis has shown that removal or alteration of VP1which is translated from an alternatively spliced message results in areduced yield of infections particles (15, 16, 38). Mutations within theVP3 coding region result in the failure to produce any single-strandedprogeny DNA or infectious particles (15, 16, 38).

The following features of AAV have made it an attractive vector for genetransfer (16). AAV vectors have been shown in vitro to stably integrateinto the cellular genome; possess a broad host range; transduce bothdividing and non dividing cells in vitro and in vive (13, 20, 30, 32)and maintain high levels of expression of the transduced genes (41).Viral particles are heat stable, resistant to solvents, detergents,changes in pH, temperature, and can be concentrated on CsCl gradients(1,2). Integration of AAV provirus is not associated with any long termnegative effects on cell growth or differentiation (3,42). The ITRs havebeen shown to be the only cis elements required for replication,packaging and integration (35) and may contain some promoter activities(14).

Initial data indicate that AAV4 is a unique member of this family. DNAhybridization data indicated a similar level of homology for AAV1-4(31). However, in contrast to the other AAVs only one ORF correspondingto the capsid proteins was identified in AAV4 and no ORF was detectedfor the Rep proteins (27).

AAV2 was originally thought to infect a wide variety of cell typesprovided the appropriate helper virus was present. Recent work has shownthat some cell lines are transduced very poorly by AAV2 (30). While thereceptor has not been completely characterized, binding studies haveindicated that it is poorly expressed on erythroid cells (26).Recombinant AAV2 transduction of CD34′, bone marrow pluripotent cells,requires a multiplicity of infection (MOI) of 10⁴ particles per cell (A.W. Nienhuis unpublished results). This suggests that transduction isoccurring by a non-specific mechanism or that the density of receptorsdisplayed on the cell surface is low compared to other cell types.

The present invention provides a vector comprising the AAV4 virus aswell as AAV4 viral particles. While AAV4 is similar to AAV2, the twoviruses are found herein to be physically and genetically distinct.These differences endow AAV4 with some unique advantages which bettersuit it as a vector for gene therapy. For example, the wt AAV4 genome islarger than AAV2, allowing for efficient encapsidation of a largerrecombinant genome. Furthermore, wt AAV4 particles have a greaterbuoyant density than AAV2 particles and therefore are more easilyseparated from contaminating helper virus and empty AAV particles thanAAV2-based particles. Additionally, in contrast to AAV1, 2, and 3, AAV4,is able to hemagglutinate human, guinea pig, and sheep erythrocytes(18).

Furthermore, as shown herein, AAV4 capsid protein, again surprisingly,is distinct from AAV2 capsid protein and exhibits different tissuetropism. AAV2 and AAV4 have been shown to be serologically distinct andthus, in a gene therapy application, AAV4 would allow for transductionof a patient who already possesses neutralizing antibodies to AAV2either as a result of natural immunological defense or from priorexposure to AAV2 vectors. Thus, the present invention, by providingthese new recombinant vectors and particles based on AAV4 provides a newand highly useful series of vectors.

SUMMARY OF THE INVENTION

The present invention provides a nucleic acid vector comprising a pairof adeno-associated virus 4 (AAV4) inverted terminal repeats and apromoter between the inverted terminal repeats.

The present invention further provides an AAV4 particle containing avector comprising a pair of AAV2 inverted terminal repeats.

Additionally, the instant invention provides an isolated nucleic acidcomprising the nucleotide sequence set forth in SEQ ID NO:1 [AAV4genome]. Furthermore, the present invention provides an isolated nucleicacid consisting essentially of the nucleotide sequence set forth in SEQID NO:1 [AAV4 genome].

The present invention provides an isolated nucleic acid encoding anadeno-associated virus 4 Rep protein. Additionally provided is anisolated AAV4 Rep protein having the amino acid sequence set forth inSEQ ID NO:2, or a unique fragment thereof. Additionally provided is anisolated AAV4 Rep protein having the amino acid sequence set forth inSEQ ID NO:8, or a unique fragment thereof. Additionally provided is anisolated AAV4 Rep protein having the amino acid sequence set forth inSEQ ID NO:9, or a unique fragment thereof. Additionally provided is anisolated AAV4 Rep protein having the amino acid sequence set forth inSEQ ID NO:10, or a unique fragment thereof. Additionally provided is anisolated AAV4 Rep protein having the amino acid sequence set forth inSEQ ID NO:11, or a unique fragment thereof.

The present invention further provides an isolated AAV4 capsid proteinhaving the amino acid sequence set forth in SEQ ID NO:4. Additionallyprovided is an isolated AAV4 capsid protein having the amino acidsequence set forth in SEQ ID NO:16. Also provided is an isolated AAV4capsid protein having the amino acid sequence set forth in SEQ ID NO:18.

The present invention additionally provides an isolated nucleic acidencoding adeno-associated virus 4 capsid protein.

The present invention further provides an AAV4 particle comprising acapsid protein consisting essentially of the amino acid sequence setforth in SEQ ID NO:4.

Additionally provided by the present invention is an isolated nucleicacid comprising an AAV4 p5 promoter.

The instant invention provides a method of screening a cell forinfectivity by AAV4 comprising contacting the cell with AAV4 anddetecting the presence of AAV4 in the cells.

The present invention further provides a method of delivering a nucleicacid to a cell comprising administering to the cell an AAV4 particlecontaining a vector comprising the nucleic acid inserted between a pairof AAV inverted terminal repeats, thereby delivering the nucleic acid tothe cell.

The present invention also provides a method of delivering a nucleicacid to a subject comprising administering to a cell from the subject anAAV4 particle comprising the nucleic acid inserted between a pair of AAVinverted terminal repeats, and returning the cell to the subject,thereby delivering the nucleic acid to the subject.

The present invention further provides a method of delivering a nucleicacid to a subject comprising administering to a cell from the subject anAAV4 particle comprising the nucleic acid inserted between a pair of AAVinverted terminal repeats, and returning the cell to the subject,thereby delivering the nucleic acid to the subject.

The present invention also provides a method of delivering a nucleicacid to a cell in a subject comprising administering to the subject anAAV4 particle comprising the nucleic acid inserted between a pair of AAVinverted terminal repeats, thereby delivering the nucleic acid to a cellin the subject.

The instant invention further provides a method of delivering a nucleicacid to a cell in a subject having antibodies to AAV2 comprisingadministering to the subject an AAV4 particle comprising the nucleicacid, thereby delivering the nucleic acid to a cell in the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic outline of AAV 4. Promoters are indicated byhorizontal arrows with their corresponding map positions indicatedabove. The polyadenylation site is indicated by a vertical arrow and thetwo open reading frames are indicated by black boxes. The splice regionis indicated by a shaded box.

FIG. 2 shows AAV4 ITR. The sequence of the ITR (SEQ ID NO: 20) is shownin the hairpin conformation. The putative Rep binding site is boxed. Thecleavage site in the trs is indicated by an arrow. Bases which differfrom the ITR of AAV2 are outlined.

FIG. 3 shows cotransduction of rAAV2 and rAAV4. Cos cells weretransduced with a constant amount of rAAV2 or rAAV4 expressing betagalactosidase and increasing amounts of rAAV2 expressing human factor IX(rAAV2FIX). For the competition the number of positive cells detected inthe cotransduced wells was divided by the number of positive cells inthe control wells (cells transduced with only rAAV2LacZ or rAAV4LacZ)and expressed as a percent of the control. This value was plottedagainst the number of particles of rAAV2FIX.

FIG. 4 shows effect of trypsin treatment on cos cell transduction. Coscell monolayers were trypsinized and diluted in complete media. Cellswere incubated with virus at an MOI of 260 and following cell attachmentthe virus was removed. As a control an equal number of cos cells wereplated and allowed to attach overnight before transduction with virusfor the same amount of time. The number of positive cells was determinedby staining 50 hrs post transduction. The data is presented as a ratioof the number of positive cells seen with the trypsinized group and thecontrol group.

DETAILED DESCRIPTION OF THE INVENTION

As used in the specification and in the claims, “a” can mean one ormore, depending upon the context in which it is used.

The present invention provides the nucleotide sequence of theadeno-associated virus 4 (AAV4) genome and vectors and particles derivedtherefrom. Specifically, the present invention provides a nucleic acidvector comprising a pair of AAV4 inverted terminal repeats (ITRs) and apromoter between the inverted terminal repeats. The AAV4 ITRs areexemplified by the nucleotide sequence set forth in SEQ ID NO:6 and SEQID NO:20; however, these sequences can have minor modifications andstill be contemplated to constitute AAV4 ITRs. The nucleic acid listedin SEQ ID NO:6 depicts the ITR in the “flip” orientation of the ITR. Thenucleic acid listed in SEQ ID NO:20 depicts the ITR in the “flop”orientation of the ITR. Minor modifications in an ITR of eitherorientation are those that will not interfere with the hairpin structureformed by the AAV4 ITR as described herein and known in the art.Furthermore, to be considered within the term “AAV4 ITRs” the nucleotidesequence must retain the Rep binding site described herein andexemplified in SEQ ID NO:6 and SEQ ID NO:20, i.e., it must retain one orboth features described herein that distinguish the AAV4 ITR from theAAV2 ITR: (1) four (rather than three as in AAV2) “GAGC” repeats and (2)in the AAV4 ITR Rep binding site the fourth nucleotide in the first two“GAGC” repeats is a T rather than a C.

The promoter can be any desired promoter, selected by knownconsiderations, such as the level of expression of a nucleic acidfunctionally linked to the promoter and the cell type in which thevector is to be used. Promoters can be an exogenous or an endogenouspromoter. Promoters can include, for example, known strong promoterssuch as SV40 or the inducible metallothionein promoter, or an AAVpromoter, such as an AAV p5 promoter. Additional examples of promotersinclude promoters derived from actin genes, immunoglobulin genes,cytomegalovirus (CMV), adenovirus, bovine papilloma virus, adenoviralpromoters, such as the adenoviral major late promoter, an inducible heatshock promoter, respiratory syncytial virus, Rous sarcomas virus (RSV),etc. Specifically, the promoter can be AAV2 p5 promoter or AAV4 p5promoter. More specifically, the AAV4 p5 promoter can be aboutnucleotides 130 to 291 of SEQ ID NO: 1. Additionally, the p5 promotermay be enhanced by nucleotides 1-130. Furthermore, smaller fragments ofp5 promoter that retain promoter activity can readily be determined bystandard procedures including, for example, constructing a series ofdeletions in the p5 promoter, linking the deletion to a reporter gene,and determining whether the reporter gene is expressed, i.e.,transcribed and/or translated.

It should be recognized that the nucleotide and amino acid sequences setforth herein may contain minor sequencing errors. Such errors in thenucleotide sequences can be corrected, for example, by using thehybridization procedure described above with various probes derived fromthe described sequences such that the coding sequence can be reisolatedand resequenced. The corresponding amino acid sequence can then becorrected accordingly.

The AAV4 vector can further comprise an exogenous nucleic acidfunctionally linked to the promoter. By “heterologous nucleic acid” ismeant that any heterologous or exogenous nucleic acid can be insertedinto the vector for transfer into a cell, tissue or organism. Thenucleic acid can encode a polypeptide or protein or an antisense RNA,for example. By “functionally linked” is meant such that the promotercan promote expression of the heterologous nucleic acid, as is known inthe art, such as appropriate orientation of the promoter relative to theheterologous nucleic acid. Furthermore, the heterologous nucleic acidpreferably has all appropriate sequences for expression of the nucleicacid, as known in the art, to functionally encode, i.e., allow thenucleic acid to be expressed. The nucleic acid can include, for example,expression control sequences, such as an enhancer, and necessaryinformation processing sites, such as ribosome binding sites, RNA splicesites, polyadenylation sites, and transcriptional terminator sequences.

The heterologous nucleic acid can encode beneficial proteins thatreplace missing or defective proteins required by the subject into whichthe vector in transferred or can encode a cytotoxic polypeptide that canbe directed, e.g., to cancer cells or other cells whose death would bebeneficial to the subject. The heterologous nucleic acid can also encodeantisense RNAs that can bind to, and thereby inactivate, mRNAs made bythe subject that encode harmful proteins. In one embodiment, antisensepolynucleotides can be produced from a heterologous expression cassettein an AAV4 viral construct where the expression cassette contains asequence that promotes cell-type specific expression (Wirak et al., EMBO10:289 (1991)). For general methods relating to antisensepolynucleotides, see Antisense RNA and DNA, D. A. Melton, Ed., ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y. (1988).

Examples of heterologous nucleic acids which can be administered to acell or subject as part of the present AAV4 vector can include, but arenot limited to the following: nucleic acids encoding therapeutic agents,such as tumor necrosis factors (TNF), such as TNF-α; interferons, suchas interferon-α, interferon-β, and interferon-γ; interleukins, such asIL-1, IL-1β, and ILs-2 through -14; GM-CSF; adenosine deaminase;cellular growth factors, such as lymphokines; soluble CD4; Factor VIII;Factor IX; T-cell receptors; LDL receptor; ApoE; ApoC; alpha-1antitrypsin; ornithine transcarbamylase (OTC); cystic fibrosistransmembrane receptor (CFTR); insulin; Fc receptors for antigen bindingdomains of antibodies, such as immunoglobulins; and antisense sequenceswhich inhibit viral replication, such as antisense sequences whichinhibit replication of hepatitis B or hepatitis non-A, non-B virus. Thenucleic acid is chosen considering several factors, including the cellto be transfected. Where the target cell is a blood cell, for example,particularly useful nucleic acids to use are those which allow the bloodcells to exert a therapeutic effect, such as a gene encoding a clottingfactor for use in treatment of hemophilia. Furthermore, the nucleic acidcan encode more than one gene product, limited only, if the nucleic acidis to be packaged in a capsid, by the size of nucleic acid that can bepackaged.

Furthermore, suitable nucleic acids can include those that, whentransferred into a primary cell, such as a blood cell, cause thetransferred cell to target a site in the body where that cell's presencewould be beneficial. For example, blood cells such as TIL cells can bemodified, such as by transfer into the cell of a Fab portion of amonoclonal antibody, to recognize a selected antigen. Another examplewould be to introduce a nucleic acid that would target a therapeuticblood cell to tumor cells. Nucleic acids useful in treating cancer cellsinclude those encoding chemotactic factors which cause an inflammatoryresponse at a specific site, thereby having a therapeutic effect.

Cells, particularly blood cells, having such nucleic acids transferredinto them can be useful in a variety of diseases, syndromes andconditions. For example, suitable nucleic acids include nucleic acidsencoding soluble CD4, used in the treatment of AIDS and α-antitrypsin,used in the treatment of emphysema caused by α-antitrypsin deficiency.Other diseases, syndromes and conditions in which such cells can beuseful include, for example, adenosine deaminase deficiency, sickle celldeficiency, brain disorders such as Alzheimer's disease, thalassemia,hemophilia, diabetes, phenylketonuria, growth disorders and heartdiseases, such as those caused by alterations in cholesterol metabolism,and defects of the immune system.

As another example, hepatocytes can be transfected with the presentvectors having useful nucleic acids to treat liver disease. For example,a nucleic acid encoding OTC can be used to transfect hepatocytes (exvivo and returned to the liver or in vivo) to treat congenitalhyperammonemia, caused by an inherited deficiency in OTC. Anotherexample is to use a nucleic acid encoding LDL to target hepatocytes exvivo or in vivo to treat inherited LDL receptor deficiency. Suchtransfected hepatocytes can also be used to treat acquired infectiousdiseases, such as diseases resulting from a viral infection. Forexample, transduced hepatocyte precursors can be used to treat viralhepatitis, such as hepatitis B and non-A, non-B hepatitis, for exampleby transducing the hepatocyte precursor with a nucleic acid encoding anantisense RNA that inhibits viral replication. Another example includestransferring a vector of the present invention having a nucleic acidencoding a protein, such as α-interferon, which can confer resistance tothe hepatitis virus.

For a procedure using transfected hepatocytes or hepatocyte precursors,hepatocyte precursors having a vector of the present inventiontransferred in can be grown in tissue culture, removed form the tissueculture vessel, and introduced to the body, such as by a surgicalmethod. In this example, the tissue would be placed directly into theliver, or into the body cavity in proximity to the liver, as in atransplant or graft. Alternatively, the cells can simply be directlyinjected into the liver, into the portal circulatory system, or into thespleen, from which the cells can be transported to the liver via thecirculatory system. Furthermore, the cells can be attached to a support,such as microcarrier beads, which can then be introduced, such as byinjection, into the peritoneal cavity. Once the cells are in the liver,by whatever means, the cells can then express the nucleic acid and/ordifferentiate into mature hepatocytes which can express the nucleicacid.

The present invention also contemplates any unique fragment of theseAAV4 nucleic acids, including the AAV4 nucleic acids set forth in SEQ IDNOs: 1, 3, 5, 6, 7, 12-15, 17 and 19. To be unique, the fragment must beof sufficient size to distinguish it from other known sequences, mostreadily determined by comparing any nucleic acid fragment to thenucleotide sequences of nucleic acids in computer databases, such asGenBank. Such comparative searches are standard in the art. Typically, aunique fragment useful as a primer or probe will be at least about 8 or10 to about 20 or 25 nucleotides in length, depending upon the specificnucleotide content of the sequence. Additionally, fragments can be, forexample, at least about 30, 40, 50, 75, 100, 200 or 500 nucleotides inlength. The nucleic acid can be single or double stranded, dependingupon the purpose for which it is intended.

The present invention further provides an AAV4 Capsid polypeptide or aunique fragment thereof. AAV4 capsid polypeptide is encoded by ORF 2 ofAAV4. Specifically, the present invention provides an AAV4 Capsidprotein comprising the amino acid sequence encoded by nucleotides2260-4467 of the nucleotide sequence set forth in SEQ ID NO:1, or aunique fragment of such protein. The present invention also provides anAAV4 Capsid protein consisting essentially of the amino acid sequenceencoded by nucleotides 2260-4467 of the nucleotide sequence set forth inSEQ ID NO:1, or a unique fragment of such protein. The present inventionfurther provides the individual AAV4 coat proteins, VP1, VP2 and VP3.Thus, the present invention provides an isolated polypeptide having theamino acid sequence set forth in SEQ ID NO:4 (VP1). The presentinvention additionally provides an isolated polypeptide having the aminoacid sequence set forth in SEQ ID NO:16 (VP2). The present inventionalso provides an isolated polypeptide having the amino acid sequence setforth in SEQ ID NO:18 (VP3). By “unique fragment thereof” is meant anysmaller polypeptide fragment encoded by any AAV4 capsid gene that is ofsufficient length to be unique to the AAV4 Capsid protein. Substitutionsand modifications of the amino acid sequence can be made as describedabove and, further, can include protein processing modifications, suchas glycosylation, to the polypeptide. However, an AAV4 Capsidpolypeptide including all three coat proteins will have at least about63% overall homology to the polypeptide encoded by nucleotides 2260-4467of the sequence set forth in SEQ ID NO: 1. The protein can have about65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% oreven 100% homology to the amino acid sequence encoded by the nucleotides2260-4467 of the sequence set forth in SEQ ID NO:1. An AAV4 VP2polypeptide can have at least about 58%, about 60%, about 70%, about80%, about 90% about 95% or about 100% homology to the amino acidsequence set forth in SEQ ID NO:16. An AAV4 VP3 polypeptide can have atleast about 60%, about 70%, about 80%, about 90% about 95% or about 100%homology to the amino acid sequence set forth in SEQ ID NO:18.

The herein described AAV4 nucleic acid vector can be encapsidated in anAAV particle. In particular, it can be encapsidated in an AAV1 particle,an AAV2 particle, an AAV3 particle, an AAV4 particle, or an AAV5particle by standard methods using the appropriate capsid proteins inthe encapsidation process, as long as the nucleic acid vector fitswithin the size limitation of the particle utilized. The encapsidationprocess itself is standard in the art.

An AAV4 particle is a viral particle comprising an AAV4 capsid protein.An AAV4 capsid polypeptide encoding the entire VP1, VP2, and VP3polypeptide can overall have at least about 63% homology to thepolypeptide having the amino acid sequence encoded by nucleotides2260-4467 set forth in SEQ ID NO:1 (AAV4 capsid protein). The capsidprotein can have about 70% homology, about 75% homology, 80% homology,85% homology, 90% homology, 95% homology, 98% homology, 99% homology, oreven 100% homology to the protein having the amino acid sequence encodedby nucleotides 2260-4467 set forth in SEQ ID NO:1. The particle can be aparticle comprising both AAV4 and AAV2 capsid protein, i.e., a chimericprotein. Variations in the amino acid sequence of the AAV4 capsidprotein are contemplated herein, as long as the resulting viral particlecomprising the AAV4 capsid remains antigenically or immunologicallydistinct from AAV2, as can be routinely determined by standard methods.Specifically, for example, ELISA and Western blots can be used todetermine whether a viral particle is antigenically or immunologicallydistinct from AAV2. Furthermore, the AAV4 viral particle preferablyretains tissue tropism distinction from AAV2, such as that exemplifiedin the examples herein, though an AAV4 chimeric particle comprising atleast one AAV4 coat protein may have a different tissue tropism fromthat of an AAV4 particle consisting only of AAV4 coat proteins.

An AAV4 particle is a viral particle comprising an AAV4 capsid protein.An AAV4 capsid polypeptide encoding the entire VP1, VP2, and VP3polypeptide can overall have at least about 63% homology to thepolypeptide having the amino acid sequence encoded by nucleotides2260-4467 set forth in SEQ ID NO:1 (AAV4 capsid protein). The capsidprotein can have about 70% homology, about 75% homology, 80% homology,85% homology, 90% homology, 95% homology, 98% homology, 99% homology, oreven 100% homology to the protein having the amino acid sequence encodedby nucleotides 2260-4467 set forth in SEQ ID NO:1. The particle can be aparticle comprising both AAV4 and AAV2 capsid protein, i.e., a chimericprotein. Variations in the amino acid sequence of the AAV4 capsidprotein are contemplated herein, as long as the resulting viral particlecomprising the AAV4 capsid remains antigenically or immunologicallydistinct from AAV2, as can be routinely determined by standard methods.Specifically, for example, ELISA and Western blots can be used todetermine whether a viral particle is antigenically or immunologicallydistinct from AAV2. Furthermore, the AAV4 viral particle preferablyretains tissue tropism distinction from AAV2, such as that exemplifiedin the examples herein, though an AAV4 chimeric particle comprising atleast one AAV4 coat protein may have a different tissue tropism fromthat of an AAV4 particle consisting only of AAV4 coat proteins.

The invention further provides an AAV4 particle containing, i.e.,encapsidating, a vector comprising a pair of AAV2 inverted terminalrepeats. The nucleotide sequence of AAV2 ITRs is known in the art.Furthermore, the particle can be a particle comprising both AAV4 andAAV2 capsid protein, i.e., a chimeric protein. The vector encapsidatedin the particle can further comprise an exogenous nucleic acid insertedbetween the inverted terminal repeats.

The present invention further provides an isolated nucleic acidcomprising the nucleotide sequence set forth in SEQ ID NO:1 (AAV4genome). This nucleic acid, or portions thereof, can be inserted intoother vectors, such as plasmids, yeast artificial chromosomes, or otherviral vectors, if desired, by standard cloning methods. The presentinvention also provides an isolated nucleic acid consisting essentiallyof the nucleotide sequence set forth in SEQ ID NO:1. The nucleotides ofSEQ ID NO:1 can have minor modifications and still be contemplated bythe present invention. For example, modifications that do not alter theamino acid encoded by any given codon (such as by modification of thethird, “wobble,” position in a codon) can readily be made, and suchalterations are known in the art. Furthermore, modifications that causea resulting neutral amino acid substitution of a similar amino acid canbe made in a coding region of the genome. Additionally, modifications asdescribed herein for the AAV4 components, such as the ITRs, the p5promoter, etc. are contemplated in this invention.

The present invention additionally provides an isolated nucleic acidthat selectively hybridizes with an isolated nucleic acid consistingessentially of the nucleotide sequence set forth in SEQ ID NO:1 (AAV4genome). The present invention further provides an isolated nucleic acidthat selectively hybridizes with an isolated nucleic acid comprising thenucleotide sequence set forth in SEQ ID NO:1 (AAV4 genome). By“selectively hybridizes” as used in the claims is meant a nucleic acidthat specifically hybridizes to the particular target nucleic acid undersufficient stringency conditions to selectively hybridize to the targetnucleic acid without significant background hybridization to a nucleicacid encoding an unrelated protein, and particularly, without detectablyhybridizing to AAV2. Thus, a nucleic acid that selectively hybridizeswith a nucleic acid of the present invention will not selectivelyhybridize under stringent conditions with a nucleic acid encoding adifferent protein, and vice versa. Therefore, nucleic acids for use, forexample, as primers and probes to detect or amplify the target nucleicacids are contemplated herein. Nucleic acid fragments that selectivelyhybridize to any given nucleic acid can be used, e.g., as primers and orprobes for further hybridization or for amplification methods (e.g.,polymerase chain reaction (PCR), ligase chain reaction (LCR)).Additionally, for example, a primer or probe can be designed thatselectively hybridizes with both AAV4 and a gene of interest carriedwithin the AAV4 vector (i.e., a chimeric nucleic acid).

Stringency of hybridization is controlled by both temperature and saltconcentration of either or both of the hybridization and washing steps.Typically, the stringency of hybridization to achieve selectivehybridization involves hybridization in high ionic strength solution(6×SSC or 6×SSPE) at a temperature that is about 12-25° C. below theT_(m) (the melting temperature at which half of the molecules dissociatefrom its partner) followed by washing at a combination of temperatureand salt concentration chosen so that the washing temperature is about5° C. to 20° C. below the T_(m) The temperature and salt conditions arereadily determined empirically in preliminary experiments in whichsamples of reference DNA immobilized on filters are hybridized to alabeled nucleic acid of interest and then washed under conditions ofdifferent stringencies. Hybridization temperatures are typically higherfor DNA-RNA and RNA-RNA hybridizations. The washing temperatures can beused as described above to achieve selective stringency, as is known inthe art. (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2ndEd., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989;Kunkel et al. Methods Enzymol. 1987:154:367, 1987). A preferablestringent hybridization condition for a DNA:DNA hybridization can be atabout 68° C. (in aqueous solution) in 6×SSC or 6×SSPE followed bywashing at 68° C. Stringency of hybridization and washing, if desired,can be reduced accordingly as homology desired is decreased, andfurther, depending upon the G-C or A-T richness of any area whereinvariability is searched for. Likewise, stringency of hybridization andwashing, if desired, can be increased accordingly as homology desired isincreased, and further, depending upon the G-C or A-T richness of anyarea wherein high homology is desired, all as known in the art.

A nucleic acid that selectively hybridizes to any portion of the AAV4genome is contemplated herein. Therefore, a nucleic acid thatselectively hybridizes to AAV4 can be of longer length than the AAV4genome, it can be about the same length as the AAV4 genome or it can beshorter than the AAV4 genome. The length of the nucleic acid is limitedon the shorter end of the size range only by its specificity forhybridization to AAV4, i.e., once it is too short, typically less thanabout 5 to 7 nucleotides in length, it will no longer bind specificallyto AAV4, but rather will hybridize to numerous background nucleic acids.Additionally contemplated by this invention is a nucleic acid that has aportion that specifically hybridizes to AAV4 and a portion thatspecifically hybridizes to a gene of interest inserted within AAV4.

The present invention further provides an isolated nucleic acid encodingan adeno-associated virus 4 Rep protein. The AAV4 Rep proteins areencoded by open reading frame (ORF) 1 of the AAV4 genome. The AAV4 Repgenes are exemplified by the nucleic acid set forth in SEQ ID NO:3 (AAV4ORF1), and include a nucleic acid consisting essentially of thenucleotide sequence set forth in SEQ ID NO:3 and a nucleic acidcomprising the nucleotide sequence set forth in SEQ ID NO:3. The presentinvention also includes a nucleic acid encoding the amino acid sequenceset forth in SEQ ID NO: 2 (polypeptide encoded by AAV4 ORF1). However,the present invention includes that the Rep genes nucleic acid caninclude any one, two, three, or four of the four Rep proteins, in anyorder, in such a nucleic acid. Furthermore, minor modifications arecontemplated in the nucleic acid, such as silent mutations in the codingsequences, mutations that make neutral or conservative changes in theencoded amino acid sequence, and mutations in regulatory regions that donot disrupt the expression of the gene. Examples of other minormodifications are known in the art. Further modifications can be made inthe nucleic acid, such as to disrupt or alter expression of one or moreof the Rep proteins in order to, for example, determine the effect ofsuch a disruption; such as to mutate one or more of the Rep proteins todetermine the resulting effect, etc. However, in general, a modifiednucleic acid encoding all four Rep proteins will have at least about90%, about 93%, about 95%, about 98% or 100% homology to the sequenceset forth in SEQ ID NO:3, and the Rep polypeptide encoded therein willhave overall about 93%, about 95%, about 98%, about 99% or 100% homologywith the amino acid sequence set forth in SEQ ID NO:2.

The present invention also provides an isolated nucleic acid thatselectively hybridizes with a nucleic acid consisting essentially of thenucleotide sequence set forth in SEQ ID NO:3 and an isolated nucleicacid that selectively hybridizes with a nucleic acid comprising thenucleotide sequence set forth in SEQ ID NO:3. “Selectively hybridizing”is defined elsewhere herein.

The present invention also provides each individual AAV4 Rep protein andthe nucleic acid encoding each. Thus the present invention provides thenucleic acid encoding a Rep 40 protein, and in particular an isolatednucleic acid comprising the nucleotide sequence set forth in SEQ IDNO:12, an isolated nucleic acid consisting essentially of the nucleotidesequence set forth in SEQ ID NO:12, and a nucleic acid encoding theadeno-associated virus 4 Rep protein having the amino acid sequence setforth in SEQ ID NO:8. The present invention also provides the nucleicacid encoding a Rep 52 protein, and in particular an isolated nucleicacid comprising the nucleotide sequence set forth in SEQ ID NO:13, anisolated nucleic acid consisting essentially of the nucleotide sequenceset forth in SEQ ID NO:13, and a nucleic acid encoding theadeno-associated virus 4 Rep protein having the amino acid sequence setforth in SEQ ID NO:9. The present invention further provides the nucleicacid encoding a Rep 68 protein, and in particular an isolated nucleicacid comprising the nucleotide sequence set forth in SEQ ID NO:14, anisolated nucleic acid consisting essentially of the nucleotide sequenceset forth in SEQ ID NO:14, and a nucleic acid encoding theadeno-associated virus 4 Rep protein having the amino acid sequence setforth in SEQ ID NO:10. And, further, the present invention provides thenucleic acid encoding a Rep 78 protein, and in particular an isolatednucleic acid comprising the nucleotide sequence set forth in SEQ IDNO:15, an isolated nucleic acid consisting essentially of the nucleotidesequence set forth in SEQ ID NO:15, and a nucleic acid encoding theadeno-associated virus 4 Rep protein having the amino acid sequence setforth in SEQ ID NO:11. As described elsewhere herein, these nucleicacids can have minor modifications, including silent nucleotidesubstitutions, mutations causing neutral amino acid substitutions in theencoded proteins, and mutations in control regions that do not orminimally affect the encoded amino acid sequence.

The present invention further provides a nucleic acid encoding theentire AAV4 Capsid polypeptide. Specifically, the present inventionprovides a nucleic acid having the nucleotide sequence set for thenucleotides 2260-4467 of SEQ ID NO:1. Furthermore, the present inventionprovides a nucleic acid encoding each of the three AAV4 coat proteins,VP1, VP2, and VP3. Thus, the present invention provides a nucleic acidencoding AAV4 VP1, a nucleic acid encoding AAV4 VP2, and a nucleic acidencoding AAV4 VP3. Thus, the present invention provides a nucleic acidencoding the amino acid sequence set forth in SEQ ID NO:4 (VP1); anucleic acid encoding the amino acid sequence set forth in SEQ ID NO:16(VP2), and a nucleic acid encoding the amino acid sequence set forth inSEQ ID NO:18 (VP3). The present invention also specifically provides anucleic acid comprising SEQ ID NO:5 (VP1 gene); a nucleic acidcomprising SEQ ID NO:17 (VP2 gene); and a nucleic acid comprising SEQ IDNO:19 (VP3 gene). The present invention also specifically provides anucleic acid consisting essentially of SEQ ID NO:5 (VP1 gene), a nucleicacid consisting essentially of SEQ ID NO:17 (VP2 gene), and a nucleicacid consisting essentially of SEQ ID NO:19 (VP3 gene). Furthermore, anucleic acid encoding an AAV4 capsid protein VP1 is set forth asnucleotides 2260-4467 of SEQ ID NO:1; a nucleic acid encoding an AAV4capsid protein VP2 is set forth as nucleotides 2668-4467 of SEQ ID NO:1;and a nucleic acid encoding an AAV4 capsid protein VP3 is set forth asnucleotides 2848-4467 of SEQ ID NO:1. Minor modifications in thenucleotide sequences encoding the capsid, or coat, proteins arecontemplated, as described above for other AAV4 nucleic acids.

The present invention also provides a cell containing one or more of theherein described nucleic acids, such as the AAV4 genome, AAV4 ORF1 andORF2, each AAV4 Rep protein gene, and each AAV4 capsid protein gene.Such a cell can be any desired cell and can be selected based upon theuse intended. For example, cells can include human HeLa cells, coscells, other human and mammalian cells and cell lines. Primary culturesas well as established cultures and cell lines can be used. Nucleicacids of the present invention can be delivered into cells by anyselected means, in particular depending upon the target cells. Manydelivery means are well-known in the art. For example, electroporation,calcium phosphate precipitation, microinjection, cationic or anionicliposomes, and liposomes in combination with a nuclear localizationsignal peptide for delivery to the nucleus can be utilized, as is knownin the art. Additionally, if in a viral particle, the cells can simplybe transfected with the particle by standard means known in the art forAAV transfection.

The term “polypeptide” as used herein refers to a polymer of amino acidsand includes full-length proteins and fragments thereof. Thus,“protein,” polypeptide,” and “peptide” are often used interchangeablyherein. Substitutions can be selected by known parameters to be neutral(see, e.g., Robinson W E Jr, and Mitchell W M., AIDS 4:S151-S162(1990)). As will be appreciated by those skilled in the art, theinvention also includes those polypeptides having slight variations inamino acid sequences or other properties. Such variations may arisenaturally as allelic variations (e.g., due to genetic polymorphism) ormay be produced by human intervention (e.g., by mutagenesis of clonedDNA sequences), such as induced point, deletion, insertion andsubstitution mutants. Minor changes in amino acid sequence are generallypreferred, such as conservative amino acid replacements, small internaldeletions or insertions, and additions or deletions at the ends of themolecules. Substitutions may be designed based on, for example, themodel of Dayhoff, et al. (in Atlas of Protein Sequence and Structure1978, Nat'l Biomed. Res. Found., Washington, D.C.). These modificationscan result in changes in the amino acid sequence, provide silentmutations, modify a restriction site, or provide other specificmutations.

A polypeptide of the present invention can be readily obtained by any ofseveral means. For example, polypeptide of interest can be synthesizedmechanically by standard methods. Additionally, the coding regions ofthe genes can be expressed and the resulting polypeptide isolated bystandard methods. Furthermore, an antibody specific for the resultingpolypeptide can be raised by standard methods (see, e.g., Harlow andLane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y., 1988), and the protein can be isolated from acell expressing the nucleic acid encoding the polypeptide by selectivehybridization with the antibody. This protein can be purified to theextent desired by standard methods of protein purification (see, e.g.,Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989).

Typically, to be unique, a polypeptide fragment of the present inventionwill be at least about 5 amino acids in length; however, uniquefragments can be 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 ormore amino acids in length. A unique polypeptide will typically comprisesuch a unique fragment; however, a unique polypeptide can also bedetermined by its overall homology. A unique polypeptide can be 6, 7, 8,9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more amino acids inlength. Uniqueness of a polypeptide fragment can readily be determinedby standard methods such as searches of computer databases of knownpeptide or nucleic acid sequences or by hybridization studies to thenucleic acid encoding the protein or to the protein itself, as known inthe art.

The present invention provides an isolated AAV4 Rep protein. AAV4 Reppolypeptide is encoded by ORF1 of AAV4. Specifically, the presentinvention provides an AAV4 Rep polypeptide comprising the amino acidsequence set forth in SEQ ID NO:2, or a unique fragment thereof. Thepresent invention also provides an AAV4 Rep polypeptide consistingessentially of the amino acid sequence set forth in SEQ ID NO:2, or aunique fragment thereof. Additionally, nucleotides 291-2306 of the AAV4genome, which genome is set forth in SEQ ID NO:1, encode the AAV4 Reppolypeptide. The present invention also provides each AAV4 Rep protein.Thus the present invention provides AAV4 Rep 40, or a unique fragmentthereof. The present invention particularly provides Rep 40 having theamino acid sequence set forth in SEQ ID NO:8. The present inventionprovides AAV4 Rep 52, or a unique fragment thereof. The presentinvention particularly provides Rep 52 having the amino acid sequenceset forth in SEQ ID NO:9. The present invention provides AAV4 Rep 68, ora unique fragment thereof. The present invention particularly providesRep 68 having the amino acid sequence set forth in SEQ ID NO:10. Thepresent invention provides AAV4 Rep 78, or a unique fragment thereof.The present invention particularly provides Rep 78 having the amino acidsequence set forth in SEQ ID NO:11. By “unique fragment thereof” ismeant any smaller polypeptide fragment encoded by AAV rep gene that isof sufficient length to be unique to the Rep polypeptide. Substitutionsand modifications of the amino acid sequence can be made as describedabove and, further, can include protein processing modifications, suchas glycosylation, to the polypeptide. However, a polypeptide includingall four Rep proteins will encode a polypeptide having at least about91% overall homology to the sequence set forth in SEQ ID NO:2, and itcan have about 93%, about 95%, about 98%, about 99% or 100% homologywith the amino acid sequence set forth in SEQ ID NO:2.

The present invention further provides an AAV4 Capsid polypeptide or aunique fragment thereof. AAV4 capsid polypeptide is encoded by ORF 2 ofAAV4. Specifically, the present invention provides an AAV4 Capsidprotein comprising the amino acid sequence encoded by nucleotides2260-4467_of the nucleotide sequence set forth in SEQ ID NO:1, or aunique fragment of such protein. The present invention also provides anAAV4 Capsid protein consisting essentially of the amino acid sequenceencoded by nucleotides 2260-4467 of the nucleotide sequence set forth inSEQ ID NO:1, or a unique fragment of such protein. The present inventionfurther provides the individual AAV4 coat proteins, VP1, VP2 and VP3.Thus, the present invention provides an isolated polypeptide having theamino acid sequence set forth in SEQ ID NO:4 (VP1). The presentinvention additionally provides an isolated polypeptide having the aminoacid sequence set forth in SEQ ID NO:16 (VP2). The present inventionalso provides an isolated polypeptide having the amino acid sequence setforth in SEQ ID NO:18 (VP3). By “unique fragment thereof” is meant anysmaller polypeptide fragment encoded by any AAV4 capsid gene that is ofsufficient length to be unique to the AAV4 Capsid protein. Substitutionsand modifications of the amino acid sequence can be made as describedabove and, further, can include protein processing modifications, suchas glycosylation, to the polypeptide. However, an AAV4 Capsidpolypeptide including all three coat proteins will have at least about63% overall homology to the polypeptide encoded by nucleotides2260-4467_of the sequence set forth in SEQ ID NO: 1. The protein canhave about 65%, about 70%, about 75%, about 80%, about 85%, about 90%,about 95% or even 100% homology to the amino acid sequence encoded bythe nucleotides 2260-4467 of the sequence set forth in SEQ ID NO:4. AnAAV4 VP2 polypeptide can have at least about 58%, about 60%, about 70%,about 80%0/, about 90% about 95% or about 100% homology to the aminoacid sequence set forth in SEQ ID NO:16. An AAV4 VP3 polypeptide canhave at least about 60%, about 70%, about 80%, about 90% about 95% orabout 100% homology to the amino acid sequence set forth in SEQ IDNO:18.

The present invention further provides an isolated antibody thatspecifically binds AAV4 Rep protein. Also provided is an isolatedantibody that specifically binds the AAV4 Rep protein having the aminoacid sequence set forth in SEQ ID NO:2, or that specifically binds aunique fragment thereof. Clearly, any given antibody can recognize andbind one of a number of possible epitopes present in the polypeptide;thus only a unique portion of a polypeptide (having the epitope) mayneed to be present in an assay to determine if the antibody specificallybinds the polypeptide.

The present invention additionally provides an isolated antibody thatspecifically binds any adeno-associated virus 4 Capsid protein or thepolypeptide comprising all three AAV4 coat proteins. Also provided is anisolated antibody that specifically binds the AAV4 Capsid protein havingthe amino acid sequence set forth in SEQ ID NO:4, or that specificallybinds a unique fragment thereof. The present invention further providesan isolated antibody that specifically binds the AAV4 Capsid proteinhaving the amino acid sequence set forth in SEQ ID NO:16, or thatspecifically binds a unique fragment thereof. The invention additionallyprovides an isolated antibody that specifically binds the AAV4 Capsidprotein having the amino acid sequence set forth in SEQ ID NO:18, orthat specifically binds a unique fragment thereof. Again, any givenantibody can recognize and bind one of a number of possible epitopespresent in the polypeptide; thus only a unique portion of a polypeptide(having the epitope) may need to be present in an assay to determine ifthe antibody specifically binds the polypeptide.

The antibody can be a component of a composition that comprises anantibody that specifically binds the AAV4 protein. The composition canfurther comprise, e.g., serum, serum-free medium, or a pharmaceuticallyacceptable carrier such as physiological saline, etc.

By “an antibody that specifically binds” an AAV4 polypeptide or proteinis meant an antibody that selectively binds to an epitope on any portionof the AAV4 peptide such that the antibody selectively binds to the AAV4polypeptide, i.e., such that the antibody binds specifically to thecorresponding AAV4 polypeptide without significant background. Specificbinding by an antibody further means that the antibody can be used toselectively remove the target polypeptide from a sample comprising thepolypeptide or and can readily be determined by radioimmunoassay (RIA),bioassay, or enzyme-linked immunosorbant (ELISA) technology. An ELISAmethod effective for the detection of the specific antibody-antigenbinding can, for example, be as follows: (1) bind the antibody to asubstrate; (2) contact the bound antibody with a sample containing theantigen; (3) contact the above with a secondary antibody bound to adetectable moiety (e.g., horseradish peroxidase enzyme or alkalinephosphatase enzyme); (4) contact the above with the substrate for theenzyme; (5) contact the above with a color reagent; (6) observe thecolor change.

An antibody can include antibody fragments such as Fab fragments whichretain the binding activity. Antibodies can be made as described in,e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y. (1988). Briefly, purifiedantigen can be injected into an animal in an amount and in intervalssufficient to elicit an immune response. Antibodies can either bepurified directly, or spleen cells can be obtained from the animal. Thecells are then fused with an immortal cell line and screened forantibody secretion. Individual hybridomas are then propagated asindividual clones serving as a source for a particular monoclonalantibody.

The present invention additionally provides a method of screening a cellfor infectivity by AAV4 comprising contacting the cell with AAV4 anddetecting the presence of AAV4 in the cells. AAV4 particles can bedetected using any standard physical or biochemical methods. Forexample, physical methods that can be used for this detection include 1)polymerase chain reaction (PCR) for viral DNA or RNA, 2) directhybridization with labeled probes, 3) antibody directed against theviral structural or non-structural proteins. Catalytic methods of viraldetection include, but are not limited to, detection of site and strandspecific DNA nicking activity of Rep proteins or replication of an AAVorigin-containing substrate. Additional detection methods are outlinedin Fields, Virology, Raven Press, New York, N.Y. 1996.

For screening a cell for infectivity by AAV4 wherein the presence ofAAV4 in the cells is determined by nucleic acid hybridization methods, anucleic acid probe for such detection can comprise, for example, aunique fragment of any of the AAV4 nucleic acids provided herein. Theuniqueness of any nucleic acid probe can readily be determined asdescribed herein for unique nucleic acids. The nucleic acid can be, forexample, the nucleic acid whose nucleotide sequence is set forth in SEQID NO: 1, 3, 5, 6, 7, 12, 13, 14, 15, 17 or 19, or a unique fragmentthereof.

The present invention includes a method of determining the suitabilityof an AAV4 vector for administration to a subject comprisingadministering to an antibody-containing sample from the subject anantigenic fragment of an isolated AAV4 capsid protein, and detecting anantibody-antigen reaction in the sample, the presence of a reactionindicating the AAV4 vector to be unsuitable for use in the subject. TheAAV4 capsid protein from which an antigenic fragment is selected canhave the amino acid sequence set forth in SEQ ID NO:4. An immunogenicfragment of an isolated AAV4 capsid protein can also be used in thesemethods. The AAV4 capsid protein from which an antigenic fragment isselected can have the amino acid sequence set forth in SEQ ID NO:17. TheAAV4 capsid protein from which an antigenic fragment is selected canhave the amino acid sequence set forth in SEQ ID NO:19.

Alternatively, or additionally, an antigenic fragment of an isolatedAAV4 Rep protein can be utilized in this determination method. Animmunogenic fragment of an isolated AAV4 Rep protein can also be used inthese methods. Thus the present invention further provides a method ofdetermining the suitability of an AAV4 vector for administration to asubject comprising administering to an antibody-containing sample fromthe subject an antigenic fragment of an AAV4 Rep protein and detectingan antibody-antigen reaction in the sample, the presence of a reactionindicating the AAV4 vector to be unsuitable for use in the subject. TheAAV4 Rep protein from which an antigenic fragment is selected can havethe amino acid sequence set forth in SEQ ID NO:2. The AAV4 Rep proteinfrom which an antigenic fragment is selected can have the amino acidsequence set forth in SEQ ID NO:8. The AAV4 Rep protein from which anantigenic fragment is selected can have the amino acid sequence setforth in SEQ ID NO:9. The AAV4 Rep protein from which an antigenicfragment is selected can have the amino acid sequence set forth in SEQID NO:10. The AAV4 Rep protein from which an antigenic fragment isselected can have the amino acid sequence set forth in SEQ ID NO:11.

An antigenic or immunoreactive fragment is typically an amino acidsequence of at least about 5 consecutive amino acids, and it can bederived from the AAV4 polypeptide amino acid sequence. An antigenicfragment is any fragment unique to the AAV4 protein, as describedherein, against which an AAV4-specific antibody can be raised, bystandard methods. Thus, the resulting antibody-antigen reaction shouldbe specific for AAV4.

The AAV4 polypeptide fragments can be analyzed to determine theirantigenicity, immunogenicity and/or specificity. Briefly, variousconcentrations of a putative immunogenically specific fragment areprepared and administered to a subject and the immunological response(e.g., the production of antibodies or cell mediated immunity) of ananimal to each concentration is determined. The amounts of antigenadministered depend on the subject, e.g. a human, rabbit or a guineapig, the condition of the subject, the size of the subject, etc.Thereafter an animal so inoculated with the antigen can be exposed tothe AAV4 viral particle or AAV4 protein to test the immunoreactivity orthe antigenicity of the specific immunogenic fragment. The specificityof a putative antigenic or immunogenic fragment can be ascertained bytesting sera, other fluids or lymphocytes from the inoculated animal forcross reactivity with other closely related viruses, such as AAV1, AAV2,AAV3 and AAV5.

As will be recognized by those skilled in the art, numerous types ofimmunoassays are available for use in the present invention to detectbinding between an antibody and an AAV4 polypeptide of this invention.For instance, direct and indirect binding assays, competitive assays,sandwich assays, and the like, as are generally described in, e.g., U.S.Pat. Nos. 4,642,285; 4,376,110; 4,016,043; 3,879,262; 3,852,157;3,850,752; 3,839,153; 3,791,932; and Harlow and Lane, Antibodies, ALaboratory Manual, Cold Spring Harbor Publications, N.Y. (1988). Forexample, enzyme immunoassays such as immunofluorescence assays (IFA),enzyme linked immunosorbent assays (ELISA) and immunoblotting can bereadily adapted to accomplish the detection of the antibody. An ELISAmethod effective for the detection of the antibody bound to the antigencan, for example, be as follows: (1) bind the antigen to a substrate;(2) contact the bound antigen with a fluid or tissue sample containingthe antibody; (3) contact the above with a secondary antibody specificfor the antigen and bound to a detectable moiety (e.g., horseradishperoxidase enzyme or alkaline phosphatase enzyme); (4) contact the abovewith the substrate for the enzyme; (5) contact the above with a colorreagent; (6) observe color change.

The antibody-containing sample of this method can comprise anybiological sample which would contain the antibody or a cell containingthe antibody, such as blood, plasma, serum, bone marrow, saliva andurine.

By the “suitability of an AAV4 vector for administration to a subject”is meant a determination of whether the AAV4 vector will elicit aneutralizing immune response upon administration to a particularsubject. A vector that does not elicit a significant immune response isa potentially suitable vector, whereas a vector that elicits asignificant, neutralizing immune response is thus indicated to beunsuitable for use in that subject. Significance of any detectableimmune response is a standard parameter understood by the skilledartisan in the field. For example, one can incubate the subject's serumwith the virus, then determine whether that virus retains its ability totransduce cells in culture. If such virus cannot transduce cells inculture, the vector likely has elicited a significant immune response.

The present method further provides a method of delivering a nucleicacid to a cell comprising administering to the cell an AAV4 particlecontaining a vector comprising the nucleic acid inserted between a pairof AAV inverted terminal repeats, thereby delivering the nucleic acid tothe cell. Administration to the cell can be accomplished by any means,including simply contacting the particle, optionally contained in adesired liquid such as tissue culture medium, or a buffered salinesolution, with the cells. The particle can be allowed to remain incontact with the cells for any desired length of time, and typically theparticle is administered and allowed to remain indefinitely. For such invitro methods, the virus can be administered to the cell by standardviral transduction methods, as known in the art and as exemplifiedherein. Titers of virus to administer can vary, particularly dependingupon the cell type, but will be typical of that used for AAVtransduction in general. Additionally the titers used to transduce theparticular cells in the present examples can be utilized. The cells caninclude any desired cell, such as the following cells and cells derivedfrom the following tissues, in humans as well as other mammals, such asprimates, horse, sheep, goat, pig, dog, rat, and mouse: Adipocytes,Adenocyte, Adrenal cortex, Amnion, Aorta, Ascites, Astrocyte, Bladder,Bone, Bone marrow, Brain, Breast, Bronchus, Cardiac muscle, Cecum,Cervix, Chorion, Colon, Conjunctiva, Connective tissue, Cornea, Dermis,Duodenum, Endometrium, Endothelium, Epithelial tissue, Epidermis,Esophagus, Eye, Fascia, Fibroblasts, Foreskin, Gastric, Glial cells,Glioblast, Gonad, Hepatic cells, Histocyte, Ileum, Intestine, smallIntestine, Jejunum, Keratinocytes, Kidney, Larynx, Leukocytes, Lipocyte,Liver, Lung, Lymph node, Lymphoblast, Lymphocytes, Macrophages, Mammaryalveolar nodule, Mammary gland, Mastocyte, Maxilla, Melanocytes,Monocytes, Mouth, Myelin, Nervous tissue, Neuroblast, Neurons,Neuroglia, Osteoblasts, Osteogenic cells, Ovary, Palate, Pancreas,Papilloma, Peritoneum, Pituicytes, Pharynx, Placenta, Plasma cells,Pleura, Prostate, Rectum, Salivary gland, Skeletal muscle, Skin, Smoothmuscle, Somatic, Spleen, Squamous, Stomach, Submandibular gland,Submaxillary gland, Synoviocytes, Testis, Thymus, Thyroid, Trabeculae,Trachea, Turbinate, Umbilical cord, Ureter, and Uterus.

The AAV inverted terminal repeats in the vector for the herein describeddelivery methods can be AAV4 inverted terminal repeats. Specifically,they can comprise the nucleic acid whose nucleotide sequence is setforth in SEQ ID NO:6 or the nucleic acid whose nucleotide sequence isset forth in SEQ ID NO:20, or any fragment thereof demonstrated to haveITR functioning. The ITRs can also consist essentially of the nucleicacid whose nucleotide sequence is set forth in SEQ ID NO:6 or thenucleic acid whose nucleotide sequence is set forth in SEQ ID NO:20.Furthermore, the AAV inverted terminal repeats in the vector for theherein described nucleic acid delivery methods can also comprise AAV2inverted terminal repeats. Additionally, the AAV inverted terminalrepeats in the vector for this delivery method can also consistessentially of AAV2 inverted terminal repeats.

The present invention also includes a method of delivering a nucleicacid to a subject comprising administering to a cell from the subject anAAV4 particle comprising the nucleic acid inserted between a pair of AAVinverted terminal repeats, and returning the cell to the subject,thereby delivering the nucleic acid to the subject. The AAV ITRs can beany AAV ITRs, including AAV4 ITRs and AAV2 ITRs. For such an ex viveadministration, cells are isolated from a subject by standard meansaccording to the cell type and placed in appropriate culture medium,again according to cell type (see, e.g., ATCC catalog). Viral particlesare then contacted with the cells as described above, and the virus isallowed to transfect the cells. Cells can then be transplanted back intothe subject's body, again by means standard for the cell type and tissue(e.g., in general, U.S. Pat. No. 5,399,346; for neural cells, Dunnett,S. B. and Björklund, A., eds., Transplantation: Neural Transplantation-APractical Approach, Oxford University Press, Oxford (1992)). If desired,prior to transplantation, the cells can be studied for degree oftransfection by the virus, by known detection means and as describedherein. Cells for ex vivo transfection followed by transplantation intoa subject can be selected from those listed above, or can be any otherselected cell. Preferably, a selected cell type is examined for itscapability to be transfected by AAV4. Preferably, the selected cell willbe a cell readily transduced with AAV4 particles; however, dependingupon the application, even cells with relatively low transductionefficiencies can be useful, particularly if the cell is from a tissue ororgan in which even production of a small amount of the protein orantisense RNA encoded by the vector will be beneficial to the subject.

The present invention further provides a method of delivering a nucleicacid to a cell in a subject comprising administering to the subject anAAV4 particle comprising the nucleic acid inserted between a pair of AAVinverted terminal repeats, thereby delivering the nucleic acid to a cellin the subject. Administration can be an ex vivo administration directlyto a cell removed from a subject, such as any of the cells listed above,followed by replacement of the cell back into the subject, oradministration can be in vivo administration to a cell in the subject.For ex vivo administration, cells are isolated from a subject bystandard means according to the cell type and placed in appropriateculture medium, again according to cell type (see, e.g., ATCC catalog).Viral particles are then contacted with the cells as described above,and the virus is allowed to transfect the cells. Cells can then betransplanted back into the subject's body, again by means standard forthe cell type and tissue (e.g., for neural cells, Dunnett, S. B. andBjörklund, A., eds., Transplantation: Neural Transplantation-A PracticalApproach, Oxford University Press, Oxford (1992)). If desired, prior totransplantation, the cells can be studied for degree of transfection bythe virus, by known detection means and as described herein.

In vive administration to a human subject or an animal model can be byany of many standard means for administering viruses, depending upon thetarget organ, tissue or cell. Virus particles can be administeredorally, parenterally (e.g., intravenously), by intramuscular injection,by direct tissue or organ injection, by intraperitoneal injection,topically, transdemnally, or the like. Viral nucleic acids(non-encapsidated) can be administered, e.g., as a complex with cationicliposomes, or encapsulated in anionic liposomes. Compositions caninclude various amounts of the selected viral particle ornon-encapsidated viral nucleic acid in combination with apharmaceutically acceptable carrier and, in addition, if desired, mayinclude other medicinal agents, pharmaceutical agents, carriers,adjuvants, diluents, etc. Parental administration, if used, is generallycharacterized by injection. Injectables can be prepared in conventionalforms, either as liquid solutions or suspensions, solid forms suitablefor solution or suspension in liquid prior to injection, or asemulsions. Dosages will depend upon the mode of administration, thedisease or condition to be treated, and the individual subject'scondition, but will be that dosage typical for and used inadministration of other AAV vectors, such as AAV2 vectors. Often asingle dose can be sufficient; however, the dose can be repeated ifdesirable.

The present invention further provides a method of delivering a nucleicacid to a cell in a subject having antibodies to AAV2 comprisingadministering to the subject an AAV4 particle comprising the nucleicacid, thereby delivering the nucleic acid to a cell in the subject. Asubject that has antibodies to AAV2 can readily be determined by any ofseveral known means, such as contacting AAV2 protein(s) with anantibody-containing sample, such as blood, from a subject and detectingan antigen-antibody reaction in the sample. Delivery of the AAV4particle can be by either ex vivo or in vivo administration as hereindescribed. Thus, a subject who might have an adverse immunogenicreaction to a vector administered in an AAV2 viral particle can have adesired nucleic acid delivered using an AAV4 particle. This deliverysystem can be particularly useful for subjects who have received therapyutilizing AAV2 particles in the past and have developed antibodies toAAV2. An AAV4 regimen can now be substituted to deliver the desirednucleic acid.

STATEMENT OF UTILITY

The present invention provides recombinant vectors based on AAV4. Suchvectors may be useful for transducing erythroid progenitor cells whichis very inefficient with AAV2 based vectors. In addition to transductionof other cell types, transduction of erythroid cells would be useful forthe treatment of cancer and genetic diseases which can be corrected bybone marrow transplants using matched donors. Some examples of this typeof treatment include, but are not limited to, the introduction of atherapeutic gene such as genes encoding interferons, interleukins, tumornecrosis factors, adenosine deaminase, cellular growth factors such aslymphokines, blood coagulation factors such as factor VIII and IX,cholesterol metabolism uptake and transport protein such as EpoE and LDLreceptor, and antisense sequences to inhibit viral replication of, forexample, hepatitis or HIV.

The present invention provides a vector comprising the AAV4 virus aswell as AAV4 viral particles. While AAV4 is similar to AAV2, the twoviruses are found herein to be physically and genetically distinct.These differences endow AAV4 with some unique advantages which bettersuit it as a vector for gene therapy. For example, the wt AAV4 genome islarger than AAV2, allowing for efficient encapsidation of a largerrecombinant genome. Furthermore, wt AAV4 particles have a greaterbuoyant density than AAV2 particles and therefore are more easilyseparated from contaminating helper virus and empty AAV particles thanAAV2-based particles.

Furthermore, as shown herein, AAV4 capsid protein is distinct from AAV2capsid protein and exhibits different tissue tropism. AAV2 and AAV4 areshown herein to utilize distinct cellular receptors. AAV2 and AAV4 havebeen shown to be serologically distinct and thus, in a gene therapyapplication, AAV4 would allow for transduction of a patient who alreadypossesses neutralizing antibodies to AAV2 either as a result of naturalimmunological defense or from prior exposure to AAV2 vectors.

The present invention is more particularly described in the followingexamples which are intended as illustrative only since numerousmodifications and variations therein will be apparent to those skilledin the art.

EXAMPLES

To understand the nature of AAV4 virus and to determine its usefulnessas a vector for gene transfer, it was cloned and sequenced.

Cell Culture and Virus Propagation

Cos and HeLa cells were maintained as monolayer cultures in D10 medium(Dulbecco's modified Eagle's medium containing 10% fetal calf serum, 100μg/ml penicillin, 100 units/ml streptomycin and IX Fungizone asrecommended by the manufacturer; (GIBCO, Gaithersburg, Md., USA). Allother cell types were grown under standard conditions which have beenpreviously reported. AAV4 stocks were obtained from American TypeCulture Collection #VR-64 6.

Virus was produced as previously described for AAV2, using the Betagalactosidase vector plasmid and a helper plasmid containing the AAV4Rep and Cap genes (9). The helper plasmid was constructed in such a wayas not to allow any homologous sequence between the helper and vectorplasmids. This step was taken to minimize the potential for wild-type(wt) particle formation by homologous recombination.

Virus was isolated from 5×10⁷ cos cells by CsCl banding (9), and thedistribution of Beta galactosidase genomes across the genome wasdetermined by DNA dot blots of aliquots of gradient fractions. Themajority of packaged genomes were found in fractions with a density of1.43 which is similar to that reported for wt AAV4. This preparation ofvirus yielded 2.5×10¹¹ particles or 5000 particles/producer cell. Incomparison AAV2 isolated and CsCl banded from 8×10⁷ cells yielded1.2×10¹¹ particles or 1500 particles/producer cell. Thus, typical yieldsof rAAV4 particles/producer cell were 3-5 fold greater than that ofrAAV2 particles.

DNA Cloning and Sequencing and Analysis

In order to clone the genome of AAV4, viral lysate was amplified in coscells and then HeLa cells with the resulting viral particles isolated byCsCl banding. DNA dot blots of aliquots of the gradient fractionsindicated that peak genomes were contained in fractions with a densityof 1.41-1.45. This is very similar to the buoyant density previouslyreported for AAV4 (29). Analysis of annealed DNA obtained from thesefractions indicated a major species of 4.8 kb in length which uponrestriction analysis gave bands similar in size to those previouslyreported. Additional restriction analysis indicated the presence ofBssHII restriction sites near the ends of the DNA. Digestion with BssHIIyielded a 4.5 kb fragment which was then cloned into Bluescript SKII+and two independent clones were sequenced.

The viral sequence is now available through Genebank, accession numberU89790. DNA sequence was determined using an ABI 373A automatedsequencer and the FS dye terminator chemistry. Both strands of theplasmids were sequenced and confirmed by sequencing of a second clone.As further confirmation of the authenticity of the sequence, bases91-600 were PCR amplified from the original seed material and directlysequenced. The sequence of this region, which contains a 56 baseinsertion compared to AAV2 and 3, was found to be identical to thatderived from the cloned material. The ITR was cloned using Deep VentPolymerase (New England Biolabs) according to the manufacturesinstructions using the following primers, primer 1:5TCTAGTCTAGACTTGGCCACTCCCTCCCTCTCTGCGCGC(SEQ ID NO:21); primer 2: 51AGGCCTTAAGAGCAGTCGTCCACCACCTTGTTCC (SEQ ID NO:22). Cycling conditionswere 97° C. 20 sec, 65° C. 30 sec, 75° C. 1 min for 35 rounds. Followingthe PCR reaction, the mixture was treated with XbaI and EcoRIendonucleases and the amplified band purified by agarose gelelectrophoresis. The recovered DNA fragment was ligated into BluescriptSKII+ (Stratagene) and transformed into competent Sure strain bacteria(Stratagene). The helper plasmid (pSV40oriAAV₄₋₂) used for theproduction of recombinant virus, which contains the rep and cap genes ofAAV4, was produced by PCR with Pfu polymerase (Stratagene) according tothe manufactures instructions. The amplified sequence, nt 216-4440, wasligated into a plasmid that contains the SV40 origin of replicationpreviously described (9, 10). Cycling conditions were 95° C. 30 sec, 55°C. 30 sec, 72° C. 3 min for 20 rounds. The final clone was confirmed bysequencing. The (βgal reporter vector has been described previously (9,10).

Sequencing of this fragment revealed two open reading frames (ORF)instead of only one as previously suggested. In addition to thepreviously identified Capsid ORF in the right-hand side of the genome,an additional ORF is present on the left-hand side. Computer analysisindicated that the left-hand ORF has a high degree of homology to theRep gene of AAV2. At the amino acid level the ORF is 90% identical tothat of AAV2 with only 5% of the changes being non-conserved (SEQ IDNO:2). In contrast, the right ORF is only 62% identical at the aminoacid level when compared to the corrected AAV2 sequence. While theinternal start site of VP2 appears to be conserved, the start site forVP3 is in the middle of one of the two blocks of divergent sequence. Thesecond divergent block is in the middle of VP3. By using threedimensional structure analysis of the canine parvovirus and computeraided sequence comparisons, regions of AAV2 which might be exposed onthe surface of the virus have been identified. Comparison of the AAV2and AAV4 sequences indicates that these regions are not well conservedbetween the two viruses and suggests altered tissue tropism for the twoviruses.

Comparison of the p5 promoter region of the two viruses shows a highdegree of conservation of known functional elements (SEQ ID NO:7).Initial work by Chang et al. identified two YY1 binding sites at −60 and+1 and a TATA Box at −30 which are all conserved between AAV2 and AAV4(4). A binding site for the Rep has been identified in the p5 promoterat −17 and is also conserved (24). The only divergence between the twoviruses in this region appears to be in the sequence surrounding theseelements. AAV4 also contains an additional 56 bases in this regionbetween the p5 promoter and the TRS (nt 209-269). Based on itspositioning in the viral genome and efficient use of the limited genomespace, this sequence may possess some promoter activity or be involvedin rescue, replication or packaging of the virus.

The inverted terminal repeats were cloned by PCR using a probe derivedfrom the terminal resolution site (TRS) of the BssHI fragment and aprimer in the Rep ORF. The TRS is a sequence at the end of the stem ofthe ITR and the reverse compliment of TRS sequence was contained withinthe BssHll fragment. The resulting fragments were cloned and found tocontain a number of sequence changes compared to AAV2. However, thesechanges were found to be complementary and did not affect the ability ofthis region to fold into a hairpin structure (FIG. 2). While the TRSsite was conserved between AAV2 and AAV4 the Rep binding site containedtwo alterations which expand the binding site from 3 GAGC repeats to 4.The first two repeats in AAV4 both contain a T in the fourth positioninstead of a C. This type of repeat is present in the p5 promoter and ispresent in the consensus sequence that has been proposed for Rep binding(10) and its expansion may affect its affinity for Rep. Methylationinterference data has suggested the importance of the CITTG motif foundat the tip of one palindrome in Rep binding with the underlined Tresidues clearly affecting Rep binding to both the flip and flop forms.While most of this motif is conserved in AAV4 the middle T residue ischanged to a C (33).

Hemagglutination Assays

Hemagglutination was measured essentially as described previously (18).Serial two fold dilutions of virus in Veronal-buffered saline were mixedwith an equal volume of 0.4% human erythrocytes (type 0) in plastic Ubottom 96 well plates. The reaction was complete after a 2 hr incubationat 8° C. HA units (HAU) are defined as the reciprocal of the dilutioncausing 50% hemagglutination.

The results show that both the wild type and recombinant AAV4 virusescan hemagglutinate human red blood cells (RBCS) with HA titers ofapproximately 1024 HAU/μl and 512 HAU/μl respectively. No HA activitywas detected with AAV type 3 or recombinant AAV type 2 as well as thehelper adenovirus. If the temperature was raised to 22° C., HA activitydecreased 32-fold. Comparison of the viral particle number per RBC atthe end point dilution indicated that approximately 1-10 particles perRBC were required for hemagglutination. This value is similar to thatpreviously reported (18).

Tissue Tropism Analysis

The sequence divergence in the capsid proteins ORF which are predictedto be exposed on the surface of the virus may result in an alteredbinding specificity for AAV4 compared to AAV2. Very little is knownabout the tissue tropism of any dependovirus. While it had been shown tohemagglutinate human, guinea pig, and sheep erythrocytes, it is thoughtto be exclusively a simian virus (18). Therefore, to examine AAV4 tissuetropism and its species specificity, recombinant AAV4 particles whichcontained the gene for nuclear localized Beta galactosidase wereconstructed. Because of the similarity in genetic organization of AAV4and AAV2, it was determined whether AAV4 particles could be constructedcontaining a recombinant genome. Furthermore, because of the structuralsimilarities of the AAV type 2 and type 4 ITRs, a genome containing AAV2ITRs which had been previously described was used.

Tissue Tropism Analysis 1.

To study AAV transduction, a variety of cell lines were transduced with5 fold serial dilutions of either recombinant AAV2 or AAV4 particlesexpressing the gene for nuclear localized Beta galactosidase activity(Table 1). Approximately 4×10⁴ cells were exposed to virus in 0.5 mlserum free media for 1 hour and then 1 ml of the appropriate completemedia was added and the cells were incubated for 48-60 hours. The cellswere then fixed and stained for β-galactosidase activity with5-Bromo-4-Chloro-3-Indolyl-β-D-galactopyranoside (Xgal) (ICNBiomedicals) (36). Biological titers were determined by counting thenumber of positive cells in the different dilutions using a calibratedmicroscope ocular (3.1 mm²) then multiplying by the area of the well andthe dilution of the virus. Typically dilutions which gave 1-10 positivecells per field (100-1000 positive cells per 2 cm well) were used fortiter determination. Titers were determined by the average number ofcells in a minimum of 10 fields/well.

To examine difference in tissue tropism, a number of cell lines weretransduced with serial dilutions of either AAV4 or AAV2 and thebiological titers determined. As shown in Table 1, when Cos cells weretransduced with a similar number of viral particles, a similar level oftransduction was observed with AAV2 and AAV4. However, other cell linesexhibited differential transducibility by AAV2 or AAV4. Transduction ofthe human colon adenocarcinoma cell line SW480 with AAV2 was over 100times higher than that obtained with AAV4. Furthermore, both vectorstransduced SW1116, SW1463 and NIH3T3 cells relatively poorly.

TABLE 1 Cell type AAV2 AAV4 Cos 4.5 × 10⁷ 1.9 × 10⁷  SW 480 3.8 × 10⁶2.8 × 10⁴  SW 1116 5.2 × 10⁴ 8 × 10³ SW1463 8.8 × 10⁴ 8 × 10³ SW620 8.8× 10⁴ ND NIH 3T3  2 × 10⁴ 8 × 10³

Tissue Tropism Analysis 2.

A. Transduction of Cells.

Exponentially growing cells (2×10⁴) were plated in each well of a 12well plate and transduced with serial dilutions of virus in 200 μl ofmedium for 1 hr. After this period, 800 μl of additional medium wasadded and incubated for 48 hrs. The cells were then fixed and stainedfor β-galactosidase activity overnight with5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (Xgal) (ICNBiomedicals) (36). No endogenous β-galactosidase activity was visibleafter 24 hr incubation in Xgal solution. Infectious titers weredetermined by counting the number of positive cells in the differentdilutions using a calibrated microscope ocular (diameter 3.1 mm²) thenmultiplying by the area of the well and the dilution of the virus.Titers were determined by the average number of cells in a minimum of 10fields/well.

As shown in Table 2, cos cells transduced with equivalent amounts ofrAAV2 and rAAV4 particles resulted in similar transduction levels.However, other cell lines exhibited differential transducibility.Transduction of the human colon adenocarcinoma cell line, SW480, withrAAV2 was 60 times higher than that obtained with rAAV4. HeLa and SW620cells were also transduced more efficiently with rAAV2 than rAAV4. Incontrast, transduction of primary rat brain cultures exhibited a greatertransduction of glial and neuronal cells with rAAV4 compared to rAAV2.Because of the heterogeneous nature of the cell population in the ratbrain cultures, only relative transduction efficiencies are reported(Table 2).

As a control for adenovirus contamination of the viral preparations cosand HeLa cells were coinfected with RAAV and adenovirus then stainedafter 24 hr. While the titer of rAAV2 increased in the presence of Ad inboth cos and HeLa, adenovirus only increased the titer in the cos cellstransduced with rAAV4 and not the HeLa cells, suggesting the differencein transduction efficiencies is not the result of adenoviruscontamination. Furthermore, both vectors transduced SW1116, SW1463,NIH3T3 and monkey fibroblasts FL2 cells very poorly. Thus AAV4 mayutilize a cellular receptor distinct from that of AAV2.

TABLE 2 CELL TYPE AAV2 AAV4 Primary Rat Brain 1  4.3□ 0.7 cos 4.2 ×10⁷□4.6 × 10⁶ 2.2 × 10⁷□2.5 × 10⁶ SW 480 7.75 × 10⁶□1.7 × 10⁶  1.3 ×10⁵□6.8 × 10⁴ HeLa 2.1 × 10⁷□1 × 10⁶  1.3 × 10⁶□1 × 10⁵  SW620 1.2 ×10⁵□3.9 × 10⁴ 4 × 10⁴ KLEB 1.2 × 10⁵□3.5 × 10⁴  9 × 10⁴□1.4 × 10⁴ HB 5.6× 10⁵□2 × 10⁵  3.8 × 10⁴□1.8 × 10⁴ SW1116 5.2 × 10⁴ 8 × 10³ SW1463 8.8 ×10⁴ 8 × 10³ NIH 3T3   3 × 10³ 2 × 10³

B. Competition Assay.

Cos cells were plated at 2×10⁴/well in 12 well plates 12-24 hrs prior totransduction. Cells were transduced with 0.5×10⁷ particles of rAAV2 orrAAV4 (containing the LacZ gene) in 200 μl of DMEM and increasingamounts of rAAV2 containing the gene for the human coagulation factorIX. Prior to transduction the CsCl was removed from the virus bydialysis against isotonic saline. After 1 hr incubation with therecombinant virus the culture medium was supplemented with completemedium and allowed to incubate for 48-60 hrs. The cells were thenstained and counted as described above.

AAV4 utilization of a cellular receptor distinct from that of AAV2 wasfurther examined by cotransduction experiments with rAAV2 and rAAV4. Coscells were transduced with an equal number of rAAV2 or rAAV4 particlescontaining the LacZ gene and increasing amounts of rAAV2 particlescontaining the human coagulation factor IX gene (rAAV2FIX). At a 72:1ratio of rAAV2FIX:rAAV4LacZ only a two-fold effect on the level ofrAAV4LacZ transduction was obtained (FIG. 3). However this same ratio ofrAAV2FIX:rAAV2LacZ reduced the transduction efficiency of rAAV2LacZapproximately 10 fold. Comparison of the 50% inhibition points for thetwo viruses indicated a 7 fold difference in sensitivity.

C. Trypsinization of Cells.

An 80% confluent monolayer of cos cells (1×10⁷) was treated with 0.05%trypsin/0.02% versene solution (Biofluids) for 3-5 min at 37° C.Following detachment the trypsin was inactivated by the addition of anequal volume of media containing 10% fetal calf serum. The cells werethen further diluted to a final concentration of 1×10⁴/ml. One ml ofcells was plated in a 12 well dish and incubated with virus at amultiplicity of infection (MOI) of 260 for 1-2 hrs. Following attachmentof the cells the media containing the virus was removed, the cellswashed and fresh media was added. Control cells were plated at the sametime but were not transduced until the next day. Transduction conditionswere done as described above for the trypsinized cell group. The numberof transduced cells was determined by staining 48-60 hrs posttransduction and counted as described above.

Previous research had shown that binding and infection of AAV2 isinhibited by trypsin treatment of cells (26). Transduction of cos cellswith rAAV2lacZ gene was also inhibited by trypsin treatment prior totransduction (FIG. 4). In contrast trypsin treatment had a minimaleffect on rAAV4lacZ transduction. This result and the previouscompetition experiment are both consistent with the utilization ofdistinct cellular receptors for AAV2 and AAV4.

AAV4 is a distinct virus based on sequence analysis, physical propertiesof the virion, hemagglutination activity, and tissue tropism. Thesequence data indicates that AAV4 is a distinct virus from that of AAV2.In contrast to original reports, AAV4 contains two open reading frameswhich code for either Rep proteins or Capsid proteins. AAV4 containsadditional sequence upstream of the p5 promoter which may affectpromoter activity, packaging or particle stability. Furthermore, AAV4contains an expanded Rep binding site in its ITR which could alter itsactivity as an origin of replication or promoter. The majority of thedifferences in the Capsid proteins lies in regions which have beenproposed to be on the exterior surface of the parvovirus. These changesare most likely responsible for the lack of cross reacting antibodies,hemagglutinate activity, and the altered tissue tropism compared toAAV2. Furthermore, in contrast to previous reports AAV4 is able totransduce human as well as monkey cells.

Throughout this application, various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains.

Although the present process has been described with reference tospecific details of certain embodiments thereof, it is not intended thatsuch details should be regarded as limitations upon the scope of theinvention except as and to the extent that they are included in theaccompanying claims.

REFERENCES

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1-42. (canceled)
 43. A nucleic acid vector comprising: a) a pair ofinverted terminal repeats, each of which is capable of forming aT-shaped hairpin structure, and wherein at least one inverted terminalrepeat comprises an AAV4 Rep protein binding site and an AAV4 terminalresolution site (trs); and, b) a heterologous nucleic acid sequencebetween the terminal repeats.
 44. The nucleic acid vector of claim 43,wherein the inverted terminal repeats are AAV4 terminal repeats.
 45. Thenucleic acid vector of claim 43, wherein the inverted terminal repeatsare at least 80% identical in sequence to SEQ ID NO:6 or SEQ ID NO:20,and wherein at least one inverted terminal repeat comprises an AAV4 Repprotein binding site and an AAV4 trs.
 46. The nucleic acid vector ofclaim 43, wherein the inverted terminal repeats are at least 90%identical in sequence to SEQ ID NO:6 or SEQ ID NO:20, and wherein atleast one inverted terminal repeat comprises an AAV4 Rep protein bindingsite and an AAV4 trs.
 47. The nucleic acid vector of claim 43, whereineach of the inverted terminal repeats comprises SEQ ID NO:6 or SEQ IDNO:20.
 48. The nucleic acid vector of claim 43, wherein the heterologousnucleic acid sequence is functionally linked to a promoter.
 49. Thenucleic acid vector of claim 48, wherein the promoter is selected fromthe group consisting of an AAV promoter, an actin gene promoter, animmunoglobulin gene promoter, a cytomegalovirus (CMV) promoter, anadenovirus promoter, a bovine papilloma virus promoter, a heat shockpromoter, a respiratory syncytial virus promoter and a Rous Sarcomavirus promoter.
 50. The nucleic acid vector of claim 48, wherein thepromoter is an AAV2 or an AAV4 promoter.
 51. The nucleic acid vector ofclaim 48, wherein the promoter is an AAV2 p5 promoter or an AAV4 p5promoter.
 52. The nucleic acid vector of claim 48, wherein the promotercomprises a functional portion of SEQ ID NO:7.
 53. The nucleic acidvector of claim 43, wherein the heterologous nucleic acid sequenceencodes a therapeutic agent.
 54. The nucleic acid vector of claim 53,wherein the therapeutic agent is selected from the group consisting of acytotoxic peptide, a tumor necrosis factors (TNF), an interferon, aninterleukin, GM-CSF, adenosine deaminase, a cellular growth factor,soluble CD4, Factor VIII, Factor IX, a T-cell receptors, LDL receptor,ApoE, ApoC, alpha-1 antitrypsin, ornithine transcarbamylase (OTC),cystic fibrosis transmembrane receptor (CFTR), insulin, Fc receptors forantigen binding domains of antibodies, and an antisense RNA.
 55. Arecombinant AAV4 particle comprising a nucleic acid vector comprising:a) a pair of inverted terminal repeats, each of which is capable offorming a T-shaped hairpin structure, and wherein at least one invertedterminal repeat comprises an AAV4 Rep protein binding site and an AAV4terminal resolution site (trs); and, b) a heterologous nucleic acidsequence between the terminal repeats.
 56. The recombinant AAV4 particleof claim 55, wherein the inverted terminal repeats are AAV4 terminalrepeats.
 57. The recombinant AAV4 particle of claim 55, wherein theheterologous nucleic acid sequence is functionally linked to a promoter.58. The recombinant AAV4 particle of claim 57, wherein the promoter isselected from the group consisting of an AAV promoter, an actin genepromoter, an immunoglobulin gene promoter, a cytomegalovirus (CMV)promoter, an adenovirus promoter, a bovine papilloma virus promoter, aheat shock promoter, a respiratory syncytial virus promoter and a RousSarcoma virus (RSV) promoter.
 59. The recombinant AAV4 particle of claim55, wherein the heterologous nucleic acid sequence encodes a therapeuticagent.
 60. The recombinant AAV4 particle of claim 59, wherein thetherapeutic agent is selected from the group consisting of a cytotoxicpeptide, a tumor necrosis factors (TNF), an interferon, an interleukin,GM-CSF, adenosine deaminase, a cellular growth factor, soluble CD4,Factor VIII, Factor IX, a T-cell receptors, LDL receptor, ApoE, ApoC,alpha-1 antitrypsin, ornithine transcarbamylase (OTC), cystic fibrosistransmembrane receptor (CFTR), insulin, Fc receptors for antigen bindingdomains of antibodies, and an antisense RNA.
 61. A method for treating asubject for a disease, comprising administering to a subject in need ofsuch treatment a recombinant AAV4 particle comprising a nucleic acidvector comprising: a) a pair of inverted terminal repeats, each of whichis capable of forming a T-shaped hairpin structure, and wherein at leastone inverted terminal repeat comprises an AAV4 Rep protein binding siteand an AAV4 terminal resolution site (trs); and, b) a heterologousnucleic acid sequence between the terminal repeats; wherein theheterologous nucleic acid sequence is functionally linked to a promoter,and wherein the heterologous nucleic acid sequence encodes a therapeuticagent for treating the disease.
 62. The method of claim 61, wherein thetherapeutic agent is selected from the group consisting of a cytotoxicpeptide, a tumor necrosis factors (TNF), an interferon, an interleukin,GM-CSF, adenosine deaminase, a cellular growth factor, soluble CD4,Factor VIII, Factor IX, a T-cell receptors, LDL receptor, ApoE, ApoC,alpha-1 antitrypsin, ornithine transcarbamylase (OTC), cystic fibrosistransmembrane receptor (CFTR), insulin, Fc receptors for antigen bindingdomains of antibodies, and an antisense RNA.